An evaluation of modified RNA-interference strategies to study the function of D-amino acid oxidase (DAO) in vivo.

There is now strong evidence showing that the NMDA glutamate receptor is important for brain functions such as memory and problem solving. A naturally occurring chemical, an amino acid called D-serine, helps activate this receptor and therefore can control the way we think. However, not much is known about how D-serine levels of the brain are controlled. One molecule, called DAO, is responsible for metabolising (breaking down) D-serine, and we have found large amounts of DAO made in parts of the mouse brain (the frontal cortex and hippocampus) which are involved in thinking and memory. So, we wonder if a decrease in DAO levels in the brain increases concentrations of D-serine, which in turn leads to the activation of the NMDA receptor. Before we can test this hypothesis, however, we need to find a way of decreasing DAO levels in the mouse brain. We will develop a new technique called RNA-interference (RNAi) to decrease DAO in the hippocamus and whole brain. This approach uses molecules called small interferring RNAs (siRNAs) which interfere with the machinery in brain cells that manufacture DAO from the blue-prints or 'genes'which are kept in the DNA of each cell. Although this method is beginning to be widely used to study other genes, not a lot is known about its safety or how well the approach can decrease the levels of brain molecules. We will therefore inject different types of siRNAs into the mouse brain to see how well they decrease DAO levels, and will also check whether these molecules are poisonous or toxic to brain cells by using other established methods. We will then repeat the study on small hairpin RNAs (shRNAs) which are another type of molecule that produces RNAi. In later work we will use the safest and most powerful RNAi approach to decrease DAO levels in the brain and see if D-serine concentrations and NMDA receptor activation is increased. Overall, the study will provide information on the usefullness of the RNAi approach to study molecules important to different brain functions.

Central N-methyl-D-aspartate receptors (NMDARs) are integral to neurodevelopment, neurotoxicity and cognitive processing. The activation of the NMDAR requires the concomitant binding of glutamate and D-serine (or glycine) to the receptor complex, and in the hippocampus D-serine co-activation predominates. The catabolising enzyme, D-amino acid oxidase (DAO), has recently been suggested to be a key modulator of brain D-serine concentrations, and thus may have direct influences on NMDAR function. To begin to test this hypothesis we first propose to develop an RNA-interference (RNAi) strategy to down regulate DAO gene expression in the mouse brain. The RNAi technique is a powerful approach to examine gene function in isolated neural cells and in vivo. The appoach has the potential to provide genetically modified animals in a relatively short time, and requires only small numbers of rodents akin to pharmacological studies. The need for expensive breeding programmes such as those used in current transgenic technology is therefore precluded, making RNAi more favourable to animal welfare. Using RNAi to study brain function, however, is still in its infancy and a critical evaluation of this application is necessary to ensure its validity. We have access to small interferring RNAs (siRNAs) incorporating several lipid-based chemistries and have constructed small hairpin RNAs (shRNAs) to provide optimal RNAi in vivo. The aim of this study is therefore to test the hypothesis that DAO-siRNAs containing certain chemistries and virally delivered shRNAs produce potent DAO gene silencing in the mouse brain with minimal neurotoxic/neuropathological responses. The RNAi-trigger molecules will be injected into the hippocampus (siRNAs and shRNAs) and cerebral ventricles (siRNAs only) of the mouse, and their diffusion kinetics and toxicity will be monitored. The latter will be assessed by evaluating the expression of inflammatory and glial markers in neural tissues.